Biomedical subjects

D K Ann

Publications and source records attributed to D K Ann.

At least 19 recordsLinked to original sources

PubMed2001

Requirement for high mobility group protein HMGI-C interaction with STAT3 inhibitor PIAS3 in repression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) transcription by Ras activation in salivary epithelial cells.

Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.

Animals↗

PubMed2001

Etk/Bmx activation modulates barrier function in epithelial cells.

Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O(2)) exposure, the TER and equivalent active ion transport rate (I(eq)) were reduced to <5% of the normoxia control in Pa-4 cells, whereas both TER and I(eq) were maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of beta-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of beta-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.

Actins↗

PubMed2000

Protein-tyrosine phosphatase D1, a potential regulator and effector for Tec family kinases.

Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain. The signaling mechanisms regulating Etk kinase activity remain largely unknown. To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen. Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated. The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin. In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction. Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction. Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors. The Etk-PTPD1 interaction also increased Stat3 activation. The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation. In addition, Tec (but not Btk) kinase can also be activated by PTPD1. Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.

Animals↗

PubMed2000

Identification of a novel taxol-sensitive kinase activity associated with the cytoskeleton.

The microtubule-targeted drug, taxol, enhances assembly of alphabeta tubulin dimers into microtubules. Recent work has established that taxol also elicits diverse effects on intracellular signaling. In-gel kinase assays with myelin basic protein as substrate revealed that taxol treatment significantly (P </= 0.05) reduced the activity of a 55 kD kinase present in cytoskeletal extracts from CV-1 cells. In vitro phosphorylation of myelin basic protein by tubulin immunoprecipitates revealed a comparable activity, consistent with the association of this kinase activity with microtubules. This novel kinase activity was detected in the cytoskeletal fraction of several other cell types including 10T12 fibroblasts and PC-3 prostate carcinoma cells, but was not detected in cytoskeletal fractions from HeLa cells. This taxol-sensitive kinase activity may participate in conveying information about taxol-induced structural changes in microtubules to changes in intracellular signaling.

Animals↗

PubMed2000

Differential regulation of rat aquaporin-5 promoter/enhancer activities in lung and salivary epithelial cells.

Aquaporin-5 (AQP5) is a water channel protein that is selectively expressed in respiratory, salivary, and lacrimal tissues. In order to establish the tissue-specific transcriptional programs that underlie its lung- and salivary-specific expression, a 4.5-kilobase pair DNA fragment encompassing the 5'-flanking region of the rat AQP5 gene has been characterized in detail. A major transcription start site utilized in lung and salivary glands has been localized downstream of a TATAA-like motif. Transient transfection assays of -4.3- and -1.7-AQP5-luciferase constructs in AQP5-expressing lung (MLE-15) and salivary (Pa-4) cells and nonexpressing fibroblast (NIH3T3) and epithelial (HeLa) cells demonstrate preferential transcriptional enhancement of reporter activities in MLE-15 and Pa-4 cells. Transient transfection assays of a series of 5' --> 3' deletion constructs of -4.3-AQP5-luciferase suggest that a common salivary and lung enhancer is located between nucleotides -274 and -139, and a lung-specific enhancer is located between nucleotides -894 and -710. There is one putative lung-specific repressor located in the region of nucleotides -1003/-894 and a common lung and salivary repressor located at nucleotides -503/-385. Moreover, 3' --> 5' deletions up to -171 and -127 base pairs almost abolish transcriptional activation in salivary and lung cells, respectively. Together, our findings indicate that the combination of enhancer/repressor elements within the proximal 5'-flanking region of rat AQP5 gene dictates its restricted expression in both lung and salivary cells.

3T3 Cells↗

PubMed2000

Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways.

The amiloride-sensitive epithelial Na(+) channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the alpha-subunit of ENaC, alpha-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H(2)O(2) modulates alpha-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and site-directed mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at -1334 to -1306 base pairs of the alpha-ENaC 5'-flanking region, is the major enhancer for the stimulated alpha-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress alpha-ENaC expression. Consistent with our hypothesis, exogenous H(2)O(2)-mediated repression of alpha-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signal-regulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H(2)O(2) on alpha-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the alpha-ENaC GRE in the presence of exogenous H(2)O(2). Taken together, we hypothesize that oxidative stress impairs Na(+) transport activity by inhibiting dexamethasone-dependent alpha-ENaC GRE activation via both ERK-dependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/dexamethasone effect on alpha-ENaC expression in lung and other tight epithelia.

Butadienes↗

PubMed2000

Salivary cellular signaling and gene regulation.

Protein tyrosine kinase and protein serine kinase activation has been implicated in the regulation of salivary cell proliferation and differentiation. Aberrant expression and alterations of certain tyrosine or serine kinases, such as Raf or erbB2, are known to trigger salivary tumor development (Li et al., 1997; Cho et al., 1999). It has been estimated that there are about 1000 to 2000 protein kinases in the mammalian genome, with 100 to 200 of them (i.e., 10%) being tyrosine kinase (Hanks and Hunter, 1995). At present, there are approximately 85 different tyrosine kinases identified in the GenBank database. Based on the relatively slow rate of discovery in the past few years, 100 is a better approximation of the total number of tyrosine kinases encoded by each mammalian genome. It is reasonable to assume that there are about 30 to 50 tyrosine kinases expressed in a given cell at a given differentiation/proliferation stage. This number is large enough to provide a characteristic tissue-specific tyrosine kinase expression profile, but small enough to be identified in a simple screening. The hope for tyrosine kinases as differentiation or proliferation markers rests with the possibility for the identification and characterization of a differentiation/proliferation stage-specific expression pattern in salivary cells. Several ligands that transmit signal through receptor tyrosine kinases and/or Ras/Raf/ERK kinases have been extensively studied in salivary cells. This review focuses mainly on the signaling pathways activated by Raf and Etk.

Animals↗

PubMed1999

Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells.

Etk/BMX is a non-receptor protein tyrosine kinase that requires a functional phosphatidylinositol 3-kinase via the pleckstrin homology domain to be activated by cytokine. In the present study, a conditionally active form of Etk was constructed by fusing the hormone-binding domain of estrogen receptor (ER) to an amino terminus truncated form of Etk, PHDelta1-68Etk, to generate DeltaEtk:ER. In stably transfected Pa-4DeltaEtk:ER cells, the activity of DeltaEtk:ER was stimulated within minutes by the treatment of DeltaEtk:ER stimulant, estradiol, and sustained for greater than 24 h. A robust induction in the phosphorylation of signal transducers and activators of transcription (STAT) proteins, including STAT1, STAT3, and STAT5, was accompanied with DeltaEtk:ER activation. Moreover, the conditionally activated Etk stimulated STAT1- and STAT5-dependent reporter activities by approximately 160- and approximately 15-fold, respectively, however, elicited only a modest STAT3-mediated reporter activation. Qualitatively comparable results were obtained in lung A549 cells, indicating that DeltaEtk:ER inducible system could function in an analogous fashion in different epithelial cells. Furthermore, we demonstrated that Etk activation alone augmented cyclin D1 promoter/enhancer activity via its STAT5 response element in both Pa-4DeltaEtk:ER and A549 cells. Altogether, these findings support the notion that the activation of Etk kinase is sufficient to transactivate STAT-mediated gene expression. Hence, our inducible DeltaEtk:ER system represents a novel approach to investigate the biochemical events following Etk activation and to evaluate the contribution by kinase activation of Etk alone or in conjunction with other signaling pathway(s) to the ultimate biological responses.

Animals↗

PubMed1999

Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

Biological Transport↗

PubMed1999

The gene expression of the amiloride-sensitive epithelial sodium channel alpha-subunit is regulated by antagonistic effects between glucocorticoid hormone and ras pathways in salivary epithelial cells.

The functional expression of the amiloride-sensitive epithelial sodium channel (ENaC) in select epithelia is critical for maintaining electrolyte and fluid homeostasis. Although ENaC activity is strictly dependent upon its alpha-subunit expression, little is known about the molecular mechanisms by which cells modulate alpha-ENaC gene expression. Previously, we have shown that salivary alpha-ENaC expression is transcriptionally repressed by the activation of Raf/extracellular signal-regulated protein kinase pathway. Here, this work further investigates the molecular mechanism(s) by which alpha-ENaC expression is regulated in salivary epithelial Pa-4 cells. A region located between -1.5 and -1.0 kilobase pairs of the alpha-ENaC 5'-flanking region is demonstrated to be indispensable for the maximal and Ras-repressible reporter expression. Deletional analyses using heterologous promoter constructs reveal that a DNA sequence between -1355 and -1269 base pairs functions as an enhancer conferring the high level of expression on reporter constructs, and this induction effect is inhibited by Ras pathway activation. Mutational analyses indicate that full induction and Ras-mediated repression require a glucocorticoid response element (GRE) located between -1323 and -1309 base pairs. The identified alpha-ENaC GRE encompassing sequence (-1334/-1306) is sufficient to confer glucocorticoid receptor/dexamethasone-dependent and Ras-repressible expression on both heterologous and homologous promoters. This report demon- strates for the first time that the cross-talk between glucocorticoid receptor and Ras/extracellular signal-regulated protein kinase signaling pathways results in an antagonistic effect at the transcriptional level to modulate alpha-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which alpha-ENaC expression is regulated in salivary epithelial cells.

Animals↗

PubMed1998

The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway.

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.

Animals↗

PubMed1998

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Blotting, Western↗

PubMed1998

Transcriptional regulation of salivary proline-rich protein gene expression.

Mechanisms governing gene expression and regulation in eukaryotes are remarkably complex. The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and trans-factor(s) associated with the salivary proline-rich proteins (PRPs) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol as well as by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by isoproterenol is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established.

Animals↗

PubMed1997

Oncogenic raf-1 induces the expression of non-histone chromosomal architectural protein HMGI-C via a p44/p42 mitogen-activated protein kinase-dependent pathway in salivary epithelial cells.

The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, we demonstrated that Raf-1 protein kinase activity in the mouse parotid glands was induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, we have stably transfected rat salivary epithelial Pa-4 cells with human Raf-1-estrogen receptor fusion gene (DeltaRaf-1:ER) and used mRNA differential display in search of messages induced by DeltaRaf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase resulted in a delayed and sustained increase of HMGI-C expression in the Pa-4 cells. The induction of HMGI-C mRNA level is sensitive to both the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D. The role of the extracellular signal-related kinase (ERK) signaling pathway in the HMGI-C induction was highlighted by the result that the MAP kinase kinase (MEK) inhibitor, PD 98059, blocked DeltaRaf-1:ER- and 12-O-tetradecanoylphorbol-13-acetate-stimulated HMGI-C induction. Altogether, these findings support the notion that the Raf/MEK/ERK signaling module, at least in part, regulates transcriptional activation of the chromosomal architectural protein HMGI-C.

Amino Acid Sequence↗

PubMed1997

Regulation of salivary-gland-specific gene expression.

The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and transfactor(s) governing the salivary proline-rich proteins (PRPs), amylase, and parotid secretory protein (PSP) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol and by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar-cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by Ipr is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established.

Amylases↗

PubMed1996

Involvement of nuclear orphan receptor NGFI-B in transcriptional activation of salivary-specific R15 gene by cAMP.

Proline-rich proteins (PRPs) are selectively expressed in the acinar cells of the salivary glands and are inducible by beta-agonist isoproterenol and dietary tannins. In the previous studies of rat PRP gene, R15, the 5'-flanking region up to -1.7 kilobase pairs (kb), which was thought to contain the necessary proximal regulatory elements, failed to confer the catecholamine isoproterenol and dietary tannin inducibility to the transgene expression in the salivary glands of transgenic mice. Here we analyzed distal 5'-flanking region of R15 in order to understand the mechanisms of tissue-specific and inducible gene regulation. An upstream regulatory region located between -2.4 and -1.7 kb of the R15 5'-flanking region is demonstrated to be indispensable for the salivary-specific and inducible reporter gene expression in vivo, by transgenic approach. Element(s) within the 0.7-kb (-2.4 to -1.7) region that is able to cis-activate the expression of a heterologous reporter gene expression is further elucidated by transient transfection assays in vitro. Three distinct nuclear orphan receptor NGFI-B regulatory sequences are identified within a 184-base pair (bp) minimal control region extended from -1995 to -1812 nucleotides relative to the transcription start site. When reporter gene containing this 184-bp control region and heterologous promoter was cotransfected with the NGFI-B expression construct, a transactivation that mimics the effect of cAMP is observed in the parotid cells. Finally, mutations on all three identified NGFI-B binding sites and coexpression of a dominant negative mutant construct, pCMV-NGFI-B(Delta25-195), abolish this transactivation mediated by NGFI-B. In summary, these data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites.

Animals↗

PubMed1995

Expression of human squamous cell differentiation marker, SPR1, in tracheobronchial epithelium depends on JUN and TRE motifs.

Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by co-transfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5'-flanking region contains the basal promoter activity, while the region between -162 and -96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: -141 TRE, -131 GT, -123 ETS-like, and -111 TRE-like motifs and in the enhanced protections in -141 TRE and -111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both -141 TRE and -111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the -111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.

Amino Acid Sequence↗

PubMed1995

Isolation of a cDNA encoding a novel zinc-finger protein from neuroblastoma x glioma NG108-15 cells.

A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the delta-opioid agonist D-Ala2-D-Leu5 enkephalin (DADLE) to down-regulate the delta-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein containing 14 tandemly repeated zinc fingers (Zf) with high homology to the Krüppel family of Zf proteins. NGD16-4 also contains a region homologous to the A element of the Krüppel Associated Box (KRAB) domain, a domain recently linked to transcriptional repression. Southern and Northern analyses indicate that NGD16-4 is derived from the mouse genome. Northern analysis also demonstrates that expression of NGD16-4 mRNA is much higher in several mouse neuroblastoma cell lines than in mouse brain or other tissues. Although the function of NGD16-4 is unclear, the expression pattern of NGD16-4 indicates a possible association with the processes of differentiation or transformation in the mouse.

Amino Acid Sequence↗